The research described in this proposal is focused on the development of a mass spectrometry (MS)-based technology that is designed to enable proteome-wide profiling of protease substrates. Proteases play a fundamental role in signal transduction pathways and have roles in a wide array of physiologic processes such as apoptosis, cell cycle, cancer, and differentiation. Despite the importance of proteases in signal transduction pathways, general methods to identify the targets of proteases or to identify the occurrence of proteolytic processing events in cellular signaling pathways are not available. We have developed novel chemical procedures and reagents that enable us to label proteins exclusively on their N-terminus with solid- phase, isotope-coded tags. This proposal seeks to develop and expand these techniques to develop a high- throughput MS-based proteomic technology capable of characterizing proteolytic processing in cells. The method relies on the idea that following proteolysis of a given protein, at least two products are generated, one of which has an N-terminus that is identical to the parent protein, while the other has a new N-terminus that is exposed only upon proteolysis. Thus, MS-based profiling of peptides derived from the N-terminus of proteins would provide a method to detect and identify proteolytically processed proteins. In order to develop this technology, the specific aims of this proposal are: (1) To optimize the MS of N-terminal peptides (MNP) procedure; and (2) To apply the MNP procedure to identify substrates of caspase-3 and nitric oxide (NO)- regulated proteolysis in HIV-associated lymphoma cell lines. Together, the experiments described in this proposal will result in the generation of development of a novel technology that we expect will become a fundamental tool for the analysis of proteases in cellular signaling. [unreadable] [unreadable] [unreadable]